Binding of pathological forms of prion proteins

ABSTRACT

Infective aggregating forms of proteins such as PrP, amyloid, and tau are bound selectively in the presence of the normal form protein using a polyionic binding agent such as dextran sulphate or pentosan (anionic), or polyamine compounds such as pDADMAC (cationic) under selective binding conditions including the use of n-lauroylsarcosine at mildly alkaline pH, and may then be assayed.

The present invention relates to the use of protease resistant bindingagents, typically polyionic materials such as polyanionic materialsincluding pentosan polysulphate, dextran sulphate or other polyanionicpolyglycosides or polycationic materials including polybrene,polyamidoamine dendrimer or poly(diallyldimethylammonium chloride),optionally under selective conditions to capture the pathological orrogue form of the prion protein PrP^(c) and proteins which are similarlyaggregating abnormal forms of proteins which in their normal form arenot aggregated.

Prion diseases, also referred to as transmissible spongiformencephalopathies or TSEs, have only been recognised recently. Bovinespongiform encephalopathy (BSE) was first reported in 1985. The firstcases of variant Creutzfeldt Jakob disease (vCJD) were reported in 1996.vCJD is a fatal neurodegenerative disease in humans believed to becaused by the consumption of BSE contaminated meat. The incubation timebetween infection to clinical symptoms in the human may be many years.

The only identified component of the prion, the agent causing priondiseases, is PrP^(Sc), an abnormal isoform of PrP^(C) (PrP^(Sc) is alsoreferred to as PrP^(res) and PrP^(C) also referred to as PrP^(Sen)).PrP^(Sc) has previously been regarded as being distinguished fromPrP^(C) in that it is comparatively protease resistant. Recentlyhowever, it has been published that there is a protease sensitive formof PrP^(Sc), i.e. that there is an infective form of PrP that isprotease sensitive.

It may be that the infective but protease sensitive PrP^(Sc) is able toaggregate (i.e. is aggregating in nature) but not yet aggregated or atleast only partially aggregated.

Both protease insensitive and protease sensitive forms of PrP^(Sc) andcore portions of PrP^(Sc) left after partial protease digestion (oftenreferred to in the art as Prp²⁷⁻³⁰) are referred to herein as PrP^(Sc)except where the context indicates that a specific one of these ismeant. Also, the term ‘aggregating proteins’ is used to include bothaggregated protease resistant PrP^(Sc) and similar forms of otherproteins as well as infective non-aggregated or partially aggregatedforms of PrP^(Sc) or other proteins, which may include the newlyobserved protease sensitive infective PrP^(Sc).

PrP^(C) is a GPI anchored glycoprotein of unknown function. Althoughsome other markers for prion diseases have been suggested prpSc remainsnot only an obligatory prion component, but also the only reliable anduniversally accepted marker for this family of diseases.

The currently favoured methodology for assaying for the presence ofPrP^(Sc) is to subject a sample to proteolysis with Proteinase K for aperiod sufficient to destroy PrP^(C) and then to determine the presenceof surviving PrP^(Sc) by an immunoassay using an antibody which is notselective for PrP^(Sc) in the presence of PrP^(C) (Serban et al.,Neurology, Vol. 40, No. 1, January 1990). The use of the proteasenaturally excludes the presence of an antibody as a capture agent or asa detection agent during the proteolysis step. The protease must beremoved or deactivated before the antibody can be introduced. It wouldbe desirable to avoid this limitation on the procedure.

The assay depends on the complete removal of PrP^(C) to avoid falsepositives and upon the conditions not being such as also to degradePrP^(Sc) to avoid false negatives. Such conditions of selectiveproteolysis need to be developed for each type of sample to be assayed.The sensitivity of the resulting assay is limited. For instance, inassays of bovine brain tissue, the sensitivity may only be such that areliable positive result is obtainable at about the time that the animalwould have been likely to show clinically observable symptoms of BSE.Thus, the assay has a sensitivity limit in the region of 1 μg/ml,corresponding to 10⁴-10⁵ prion infectious units.

There is a need for more sensitive and specific diagnostic tests forprion diseases. In particular, an ante-mortem test using blood or othersample types is required to assess the disease status of a particularanimal. In the absence of such a method extensive slaughtering of cattleis required once an affected animal is identified within a herd. It isagain vital that a diagnostic test is developed to screen the humanpopulation and to protect individuals from potential infection fromdonated blood, surgical procedures and organ and tissue transplants.

U.S. Pat. No. 5,977,324 and U.S. Pat. No. 6,221,614 both describemethods of binding PrP^(Sc) using phosphotungstic acid (PTA). PTA is anonspecific protein precipitant that will also bind to, and precipitate,a wide range of proteins other than PrP^(Sc). The concentration of theproteins in the sample will also greatly affect the recovery using PTA.

Plasminogen has been reported to bind PrP^(Sc) selectively with respectto PrP^(C) and was proposed for use in diagnostic assays (Fischer et al.Nature, 2000, Nov. 23, 408 (6811): 479-83). However, this method has notproved sufficiently useful in practice. Plasminogen is also identifiedin a related disclosure, U.S. 2002/0004586, as being a factor whichselectively binds PrP^(Sc).

According to U.S. Pat. No. 6,419,916 and related disclosures, thepolyamine compound Superfec™ (a branched polyamine mixture produced byheat induced degradation of a PAMAM dendrimer) and other similarbranched polyamines are capable of clearing PrP^(Sc) from cells invitro. The mechanism is unclear. It is speculated that such compoundsmay bind directly to PrP^(Sc) arranged as an amyloid with exposednegatively charged moieties and induce a conformational change underacidic conditions. It is said that the effect cannot simply involvebinding of PrP^(C) and inhibiting synthesis of PrP^(Sc) because existingPrP^(Sc) is cleared. The polyamine is found to make PrP^(Sc) proteasesensitive provided the pH is below 4. It is deduced that the polyaminesact in an acidic cell compartment in the in vitro PrP^(Sc) clearanceexperiments.

It would appear from this work that it would be speculative to concludethat such polyamines bind PrP^(Sc). A number of other possibilities areadvanced. No selectivity for the binding of PrP^(Sc) over PrP^(C) isshown or suggested. Furthermore, it cannot be deduced that any bindingthat occurs is more than transitory, just serving to alter theconformation of PrP^(Sc) so as to allow protease attack. Also, theaction of the polyamines appears to require a low pH. Our owninvestigations in fact show that such dendrimer polyamines do not bindPrP^(Sc) at such low pH.

Pentosan polysulphate (poly-b-xylose-2,3-disulphonate, PPS) is one of arange of large polysulphonated polyglycosides (PGs) (MW 8,000-12,000).Made from beechwood, it is an inexpensive compound that has been usedfor many years as an anticoagulant similar to heparin, also a PG. PGsincluding PPS and other polyanions are known to bind both PrP^(C) andrecombinant PrP (recPrP), see for instance Brimacombe D B et al, BiochemJ, 1999 Sep. 15; 342 pt 3, 605-13. PPS has accordingly been proposed asa potential therapeutic agent for preventing or treating TSE diseases.It has not however been shown to remove existing PrP^(Sc) in vivo or invitro.

In manufacture, sawdust from beechwood is extracted to produce thesoluble sugar polymer of xylose (a five member ring sugar) calledpentosan. This polymer is then subjected to a sulphation reaction usinga mixture of chlorosulphonic acid and pyridine, which results in 3 outof 4 of all the sugar ring hydroxyls having a sulphate ester added tothem. The total sulphate content is then about 50-55% by weight which ismore than in heparin, in which it is about 30-35%. The only othersimilar molecule that approaches this high degree of sulphation isdextran sulphate (40-45%). Pentosan has quite a low MW of 3.5-7.0 K.

No selectivity for binding by polyanions or polycations of PrP^(Sc) withrespect to binding of PrP^(C) has been reported. Surprisingly, we havenow established conditions under which polyionic materials bindaggregated altered proteins like PrP^(Sc) and further have establishedconditions under which such polyanions bind these abnormal forms but donot bind their non-aggregated normal forms like PrP^(C), the bindingbeing sufficiently strong and under preferred conditions sufficientlyselective to be useful in assays for the presence of the aggregatedaltered protein (e.g.PrP^(Sc)).

Accordingly, there is now provided in a first aspect of the invention, aprocess for the selective binding of an aggregating abnormal form of aprotein in the presence of the non-aggregating normal form of theprotein, comprising contacting under selective binding conditions amaterial containing both said abnormal and normal forms with a polyionicmaterial having a binding avidity for said aggregating form of theprotein as present in the sample. The binding conditions may include thepresence of a competition agent in solution, which competition agent hasa lesser binding avidity for the abnormal form of the protein than doesthe polyionic material.

The polyionic material may be in solution or may provide a surfacepresenting ionic surface groups. In the latter case, the surface may bethat of a polymer having said ionic groups covalently bonded within thestructure of the polymer or produced by modification of surface groupsof the polymer. An example of a suitable polyanionic polymer is Nafion,a perfluoronated sulphonated hydrocarbon polymer available as beads oras sheets. Polycationic polymers may also be used.

Alternatively, the surface is that of a substrate having coated thereonor bonded thereto a substance presenting said ionic groups. An exampleof a suitable polymer having such surface groups is a non-chargedplastics surface activated with maleic anhydride and derivatised withTRIS to produce surface carboxyl groups or with a polycationic material.Polycations or polyanions may instead be passively coated on polymerssuch as polystyrene.

In the case of a polyanionic material, whether used in solution orcoated on a solid surface, the polyanionic material may preferably be apolyanionic polyglycoside.

Generally, the competition agent has a lesser density of ionic groupsthan the polyionic material. Without being bound by theory, it is likelythat the findings described in detail herein are due to aggregatingabnormal forms of protein having more binding sites for interaction withionic groups than the non-aggregating normal form of the same protein. Acompetition agent having one or a few ionic groups is able to interactwith a certain avidity with either the aggregating or non-aggregatingforms of the protein but a polyionic material is able to bind theaggregated form of the protein simultaneously through many ionic groups,leading to it having a higher avidity for the aggregating than for thenon-aggregating form.

Our experimental results with infected bovine brains indicate that bothimmobilised polyanions (such as dextran sulphate) and polycations (suchas polyethyleneimine) are able to capture the abnormal form of the prionprotein PrP^(SC) in brain homogenates. The signal obtained using ananti-prion protein antibody/enzyme conjugate is approximately 3 to 5times higher for the best polycationic capture surface than for the bestpolyanionic capture surface.

In both cases the detergent Sarkosyl (N-lauroyl-sarcosine) can act as acompetition agent helpful for improving the specificity of capture ofthe abnormal protein and avoiding a signal from the normal prionprotein, when using a non-specific anti-prion protein antibody. Also,partially digesting the sample with trypsin substantially increases thesignal from PrP^(Sc) when using either polycationic or polyanioniccompounds, but has no effect on specificity (indicating that, under theconditions employed, trypsin is removing an inhibitor of polyion bindingof PrP^(Sc), rather than preferentially digesting PrP^(C), as has beenobserved for proteinase K).

It is reported in the scientific literature that PrP^(Sc) is nativelyassociated with polyanions such as heparans. It is likely that thesenegatively charged polymers are interacting with a positively chargedregion of the prPSc structure and there could be multiple interactionswith the aggregated proteins.

We propose that a polyanion such as dextran sulphate or pentosanpolysulphate is able to bind to the PrP^(Sc) structure with much higheravidity than native heparans and so can displace the endogenouscompounds. Thus, highly negatively charged polymers immobilised to asurface can capture specifically the abnormal prion protein. The normalprion protein is non-aggregating and does not have such a high affinityinteraction with endogenous heparans or with polyanions such as dextransulphate. Under the assay conditions chosen the presence of loweraffinity anions such as the detergent Sarkosyl improves the specificityof capture still further by competing with the immobilised polyanion forthe lower affinity PrP^(C) interaction sites.

In contrast we suggest that a polycation cannot displace the endogenousheparans from the PrP^(Sc) structure. We suggest that it must insteadcomplex directly with the endogenous heparan/PrP^(Sc) aggregate—formingan ionic interaction with the free negative charges on the heparan. So,in this configuration the native, intact heparan/PrP^(Sc) complex isbound tightly to the immobilised polycation whilst, in the case of animmobilised polyanion the non-native, ‘displaced’ PrP^(Sc) structure iscaptured instead. This provides an explanation for the higher signal weobtain with the best polycationic capture surfaces, in that competitionby polyanions for endogenous heparans may not be 100% efficient and sonot all of the PrP^(Sc) aggregates are bound by negatively chargedpolymers.

Anionic capture agents may be preferable when the aggregating protein isnot expected to be bound natively by native heparan, e.g. when thesample is blood or serum or the like rather than tissue.

In addition to the ionic interactions proposed, there may be additionalhydrophobic binding between other regions of the PrP^(Sc) aggregate andthe polymers employed. These will strengthen further the bindinginteractions.

“Avidity” here is used in the usual meaning of the overall bindingstrength of a molecule with many binding sites with a multivalentbinding agent and in contrast to “affinity”, being the binding strengthbetween each individual binding site and of the molecule and the bindingagent.

The competition agent if used is preferably an amino acid amide of afatty acid, such as n-lauroylsarcosine. Such materials have detergencyproperties, but in this context may well simply be acting as monovalentbinding agents via their terminal COO⁻ group or as partially polyvalentagents through the formation of micelles.

In a further aspect, the present invention provides a process for theselective binding of an aggregated abnormal form of a protein in thepresence of the non-aggregated normal form of the protein, comprisingcontacting a material containing both said abnormal and normal formswith a polyanionic polyglycoside under conditions such as to provideselective binding of said abnormal form.

In preferred embodiments of each aspect of the invention said abnormalform of a protein is PrP^(Sc) and said normal form is PrP^(C). However,the invention in all its forms is broadly applicable to the selectivebinding of abnormal aggregating forms of proteins.

Polycationic selective binding agents that can be used includepolyethyleneimines, polyamines, including poly-lysines, polyamidoamines,e.g. PAMAM dendrimers, poly-quaternary amines such aspoly(diallyldimethylammonium chloride) and1,5-dimethyl-1,5-diazaundecamethylene poly-methobromide (also known ashexadimethrine bromide or Polybrene).

The preferred polyanionic polyglycoside is a polysulphonatedpolyglycoside. However, other anionic sites such as carboxylic acidgroups or phosphate groups may be used as well or instead.

Preferably, the polysulphonated polyglycoside is pentosan polysulphate(PPS) or dextran sulphate.

Other polyanionic pentosan or dextran derivatives may be used as thepolyanionic polyglycoside.

A high level of sulphonation (or other anionic group) is preferred.

The levels of sulphonation of the carrageenins, dextrans and pentosanare high. If a low proportion of the potential sulphonation sites isactually taken up by sulphate groups then it may be found that thecompounds do not interact with the binding sites in the PrP^(Sc)selectively.

Suitable anionic selective binding agents may include:

Pentosan polysulphate (MW 3500-5000), Dextran sulphate 500 (MW 500,000),Iota-carrageenan, Lambda-carrageenin and carrageenans, e.g.Kappa-carrageenan, Heparins and heparans, Dextran sulphate 8 (MW 8,000),sulphonated polyglycosides such as fucoidan, keratin sulphate,hyaluronic acid polysulphate, colominic acid (bacterial polysialicacid), carrageenan types iii and iv, dermatan sulphate, heparansulphate, furcellaran, sulphated commerically available polysaccharidese.g. polysorbate, sizofiran, xanthan gum, starch, cellulose compounds,pectin, gastric mucin, ceratonia, agars, acacia gum, Sulphated Glycoside1, Sulphated Glycoside 2, N-acetyl-D-glucosamines, or Dermatan sulphateL-iduronic acid.

The polyionic material may be one selected to have the ability undernon-selective conditions to bind both aggregating altered or rogue formsof a protein and also the non-aggregating normal form of the protein aswell as the ability to bind the aggregating form selectively underappropriate conditions.

The desired selectivity is obtainable by suitable adjustment of thereaction conditions, particularly the presence and concentration of thecompetition agent, the pH and the detergency. Preferably therefore thepH is so selected as to provide said selective binding.

The pH is preferably from 5.6 to 9, e.g. from 7 to 9, more preferably 8to 9, e.g. from 8.2 to 8.6, especially 8.4, particularly when thedetergents described below are used. Suitable buffers include phosphatebuffers and Tris buffers.

The salt concentration is preferably not higher than 250 mM and ispreferably significantly less, e.g. not above 100 mM.

Preferably, a detergent is present which promotes said selective bindingwhether by virtue of detergency or by acting as a competition agent.

Particularly preferred for this purpose are detergents which are anamino acid amide of a fatty acid, e.g. n-lauroylsarcosine or other fattyacid sarcosines. The presence of such a detergent/competition agent isespecially, preferred when the selective binding agent is polyanionic.

Preferably the concentration of this detergent is at least 0.05% byweight, more preferably at least 0.1%, preferably at least 0.2%, e.g.0.2 to 2%, more preferably 0.5 to 1.5%, but greater amounts may be used.

Other detergents having a similar effect may be used, including CHAPS,Brij, Octyl-β-glycoside, Tween 20, Triton X-100 and Nonidet P-40. Theuse of high concentrations of sodium dodecylsulphate (SDS) is howeverundesirable. Combinations of n-lauroyl sarcosine (or similar) with otherdetergents are suitable, preferably containing 0.5 to 2%, e.g. about 1%sarcosine detergent, e.g. with 0.5 to 2%, e.g. about 1%, of one of thedetergents listed above, particularly Triton X-100 or Nonidet P-40.

We have found that the presence of trypsin, chymotrypsin, proteinase K,or another such protease can be helpful to prevent inhibition by unknownmaterials of the binding of aggregating protein to either polyanionic orpolycationic selective binding agents. This is especially the case wherethe sample contains a relatively high level of other proteins, such asis the case if a PrP^(Sc) positive brain sample is diluted with aPrP^(Sc) negative brain material. Additional matrix inhibitionprevention can be obtained by including other enzymes of a degradativenature including Dnase and collagenase.

The selective binding agent after binding to said aggregating abnormalform of the protein may be captured with an immobilised capture agentand the presence or amount of a complex formed between said selectivebinding agent and said capture agent may be determined.

Said capture agent may be a lectin (where the binding agent is suitable,e.g. is a polyglycoside) or an antibody reactive with said selectivebinding agent. Said selective binding agent may be provided with aselectively bindable tag moiety and said capture agent may then bind tosaid tag moiety.

Optionally and alternatively, the selective binding agent is immobilisedto a solid medium prior to exposure to said sample. The selectivebinding agent may be provided with a selectively bindable tag moiety andmay be immobilised to said solid medium via said tag.

Where a bindable tag moiety is present it may for instance be biotin,fluorescein, dinitrophenol, digoxygenin, or (His)6.

The selective binding agent may be immobilised directly to a solidrather than through a bindable tag. For instance PG's may be directlycoupled by covalent coupling through remaining hydroxyl groups of the PGusing solid phases derivatised with for instance epoxy or vinyl sulphonegroups.

In each aspect of the invention, whether the binding of the abnormalprotein takes place before or after the immobilisation or capture of theselective binding agent, the immobilised selective bindingagent/abnormal protein complexes are preferably subjected to a washingstep to remove normal protein to improve selectivity. The washing stepis preferably conducted using a solution containing a said competitionagent, which may be a detergent solution, which preferably againcomprises a detergent that whether by virtue of its detergency orotherwise promotes selective binding. This is preferably an amino amideof a fatty acid, e.g. n-lauroyl sarcosine or another fatty acidsarcosine. Preferably, the concentration of the sarcosine detergent inthe washing step is at least 0.05%, preferably at least 0.1%, morepreferably at least 0.2%, e.g. 0.2 to 2%, preferably 0.5 to 1.5%. Otherdetergents may also be present and the wash is preferably buffered to apH in the range of 5.6 to 8.4.

Said binding of PrP^(Sc) (or other abnormal protein) may bequalitatively or quantitatively determined by conducting an immunoassayfor PrP^(Sc) (or other aggregating protein) after separation of boundPrP^(Sc) from unbound PrP^(C) (or other normal form protein).

Also, once the aggregating form of the protein has been selectivelybound and optionally after the normal form has been removed, furtherpolyanionic material, e.g. anionic polyglycoside (suitably labelled witha tag or detectable label) may be bound to the already bound aggregatingprotein to form a sandwich (e.g. polyglycoside-aggregatedprotein-polyglycoside label) which may then be quantitated or detected.Selective binding conditions may not be necessary when carrying out thesecond part of sandwich formation.

As mentioned above, the selective binding agent may be immobilised to asolid material either before or after being contacted with the alteredprotein. Separation of the sample from the solid material may then beused to remove the normal form of the protein from the assay leavingonly the altered form for further determination.

In this context, solid support materials include not only macroscopic orhandlable materials such as microtitre plates, dipsticks and laminarflow devices, but also microbeads and superparamagnetic microbeads,which may be separated off by filtration or by magnetic capture. Biotinor other tags may be conjugated to dextran sulphate or PPS and likematerials by standard chemical methods. About one in ten of the sugarbackbone residues in PPS is a uronic acid methyl ester and this providesone route for coupling via their carboxyl residues. Other known routesfor coupling are hydroxyl (one in four is still free after thesulphation reaction), or end group reducing sugar. Biotin is aconvenient bindable tag moiety to employ for binding of the polyanionicmaterial or other selective binding agent to a solid materialderivatised with avidin or a material with avidin binding propertiessuch as steptavidin, Neutravidin or Captavidin.

Other molecules suitable for use as bindable tag moieties will includeall those which are readily conjugated to the polyionic material andwhich lend themselves to capture by a suitable capture agent. Forinstance, a molecule such as fluorescein may be conjugated to PPS orlike molecules by reacting an amino fluorescein derivative with theuronic and side chains of pentosan polyslphate in the presence ofcarbodiimide EDC(1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide) andmay be captured by a suitable readily available antibody, which mayitself be immobilised to the solid material. Other tags suitable forantibody capture in this way include dinitrophenol DNP, digoxygenin,nucleic acid or nucleic acid analog sequences, and (His) 6. Bindingagents other than antibodies may also be used, e.g. complementarynucleic acid or nucleic acid analog sequences.

Alternatively however, a capture agent may be used which selectivelybinds the polyionic material itself rather than through a tag moiety.For instance, polyglycosides may be bound by a suitable lectin or by asuitable antibody. Antibodies for binding PPS are for instance disclosedin Kongtawelert et al; J. Immunol. Methods 1990, Jan. 24; 126(1); 39-49.Standard techniques for immobilising such antibodies are well known inthe art.

Any known or in future devised method for determining the presence oramount of aggregated or aggregating altered proteins such as PrP^(SC)(without needing selectivity to exclude the normal form such as PrP^(C))can be used to determine the presence or amount of the aggregating formonce it has been selectively bound by the selective binding agent andunbound normal form protein has been separated therefrom, suitably byimmobilisation of the bound and washing away of residual unbound. Suchmethods include the known ELISA, RIA, IRMA and other forms ofimmunoassay, for instance the method embodied in the Bio-Rad Platelia™BSE Detection Kit and described in Serban et al.

Depending on the form of the assay used, it may be desired or requiredto elute the captured abnormal form protein from the selective bindingagent prior to the assay. In conducting such an elution step, thepresence of a chaotrope such as guanidine thiocynanate may be desirableat a concentration of at least 1M, preferably 2 to 6 M, e.g. 4 to 6 M.Alternative chaotropes may be used including urea.

Additionally or alternatively, a competition agent having a still higheravidity may be used to displace the protein from the selective bindingagent. Sodium dodecyl sulphate (SDS) is suitable for this and ispreferably used at a concentration of 0.5 to 1% by weight, preferablyabove 0.75%.

Other proteins that may be selectively bound and determined according tothe invention include the β-amyloid protein and tau protein which formplaques in Alzheimer's disease.

Without wishing to be bound by the following theory, it is thought thatPPS and similar molecules function in the invention by binding pairs ofnegative sulphate groups to pairs of positive amino acids (Lys and Arg)in the relevant proteins or via the proteins' polyhistidine metalbinding sites. Binding to the aggregated forms may be stronger due tothe increased number of binding sites presented by the aggregatingprotein. Suitable anionic detergents may compete more effectively forbinding with the non-aggregating form to enhance selectivity. Suitably,the selectivity obtained is such that the avidity for binding to theaggregating protein is at least three times that for the normal form,preferably at least 10:1.

In a further aspect, the invention includes a process for separatingPrP^(Sc) from PrP^(C) comprising selectively binding PrP^(Sc) to abinding agent in the presence of an amino acid amide of a fatty acid.Preferred conditions for such binding are as set out in detail above andthe bound protein may be assayed as described.

The invention will be further described and illustrated by the followingexamples making reference to the accompanying drawing in which:

FIG. 1 shows dilution curves obtained in Example 9.

EXAMPLE 1 Separation of Normal Prion from Rogue Prion Protein UsingBiotinylated Pentosan Polysulphate and Subsequent Affinity Capture

Introduction

Biotin was conjugated to pentosan polysulphate using standard chemicalmethods. The biotinylated pentosan polysulphate was allowed to bind tothe rogue prion protein in brain homogenates and after binding thepentosan polysulphate/prion complexes were captured usingstreptavidin-derivatised superparamagnetic beads. The captured rogueprion was subsequently eluted from the beads and detected using theimmuno-based Bio-Rad Platelia™ BSE Detection Kit; the latter kit isunable to differentiate the normal and rogue prion protein and will givea signal with both proteins. A bank of two BSE-infected and twouninfected bovine brains were investigated and used to demonstrate thatthe pentosan polysulphate, under the specific conditions described,could be used to specifically capture rogue prion protein from the brainhomogenates.

Method

Preparation of the Superparamagnetic Beads.

-   -   1. Just prior to use, 400 μl of streptavidin superparamagnetic        beads (Sigma-Aldrich Company Ltd., S-2415) were washed by        magnetic capture in three consecutive 1 ml volumes of TBST (50        mM Tris, 150 mM NaCl, pH 7.5, 0.05% (v/v) Tween20 [Sigma-Aldrich        Company Ltd., P-7949]).    -   2. The beads were finally resuspended in 400 μl of TEST.    -   3. 100 μl aliquots were prepared in four tubes and the liquid        removed. The beads were then ready for use.        Preparation of the Brain Homogenates.    -   1. 300-500 mg of each brain tissue was added to the grinding        tubes containing grinding beads as supplied in the BSE        Purification Kit (Bio-Rad). The liquid originally supplied in        these tubes in the kit was aspirated and discarded prior to use.    -   2. A volume of 150 mM NaCl that was calculated to generate a 50%        (w/v) brain homogenate after, homogenisation was added to each        tube.    -   3. The tubes were homogenised for 45 seconds at speed setting        6.5 on a ribolyzer (purchased from Bio-Rad).    -   4. The homogenates were diluted 1:1 with 150 mM NaCl.    -   5. 50 μl volumes of each homogenate were placed in separate        tubes.        Specific Capture of the Rogue Prion Protein    -   6. 10 μl of 20% (w/v) N-lauroylsarcosine (Sigma-Aldrich Company        Ltd., L-9150) was then added to each tube of homogenate and        mixed.    -   7. 50 μl of biotinylated pentosan polysulphate (10 μg/ml in        distilled sterile water) was then added to each tube, mixed and        incubated at room temperature for 30 minutes.    -   8. Each reaction was then added to a tube of washed streptavidin        superparamagnetic beads and incubated at room temperature for 30        mins.    -   9. The beads were then washed by magnetic capture in three 1 ml        volumes of TBST.        Elution of the Rogue Prion Protein and Immunodetection.    -   1. Finally, after the last wash, the beads from each reaction        were resuspended in 10 μl of C1 (supplied with the Bio-Rad        Platelia™ BSE Detection kit).    -   2. 5 μl of 0.2% (w/v) SDS was added to each bead suspension and        mixed.    -   3. 5 μl of 1M guanidine thiocyanate (Sigma-Aldrich Company Ltd.,        G-9277) was added to each bead suspension and mixed.    -   4. The reaction was heated at 100° C. for 5 minutes.    -   5. 100 μl of R6 (supplied with the Bio-Rad Platelia™ BSE        Detection kit) was then added and mixed.    -   6. 100 μl of each eluate was then used in the Bio-Rad Platelia™        BSE Detection kit using the protocol and reagents supplied with        this kit. Briefly, this kit involves immunocapture of normal        and/or rogue prion protein and immunodetection with a        horseradish peroxidase conjugated antibody.        Results

After performing the immunodetection in the microtiter plate-basedPlatelia™ assay the signal in each well was measured at a wavelength of450 nm using an ELISA reader. Brain homogenate used OD₄₅₀ BSE-infectedbovine 0.229 brain sample 1 BSE-infected bovine 0.208 brain sample 2Normal bovine brain 0.061 sample 1 Normal bovine brain 0.047 sample 2

The signal from the two BSE-infected brain homogenates containing rogueprion is significantly higher than in the uninfected normal brainhomogenates.

Discussion

The Bio-Rad Platelia™ BSE Detection kit cannot differentiate betweennormal or rogue prion protein. Normally, the specificity for rogue prionprotein is achieved by prior digestion of the sample with proteinase Kwhich removes the protease susceptible normal prion protein. Any rogueprion protein in the sample is more resistant to protease digestion andremains and is subsequently detected by the Platelia™ assay. In thisexperiment we have demonstrated an alternative approach to proteasedigestion of the sample. We have used defined conditions under whichbiotinylated pentosan polysulphate in solution can specifically bind tothe rogue prion protein in the sample. The rogue prion/pentosanpolysulphate complex can then be captured using streptavidinsuperparamagnetic beads. After washing, the rogue prion protein cansubsequently be eluted and detected in the immunoassay. Normal prionprotein is not captured by this protocol and is washed away and istherefore not detected in the immunoassay. We have demonstrated that byusing this technique we could correctly detect rogue prion protein intwo BSE-infected bovine brains and no signal was observed in two normalbovine brains.

EXAMPLE 2 Separation of Normal Prion from Rogue Prion Protein UsingImmobilised Biotinylated Pentosan Polysulphate

Introduction

Biotin was conjugated to pentosan polysulphate using standard chemicalmethods. The biotinylated pentosan polysulphate was used to coatstreptavidin-derivatised super-paramagnetic beads. The coated beads werethen used to specifically capture the rogue prion protein from brainhomogenates. The captured rogue prion protein was subsequently elutedfrom the beads and detected using the immuno-based Bio-Rad Platelia™ BSEDetection Kit; the latter kit is unable to differentiate the normal androgue prion protein and will give a signal with both proteins. A bank ofthree BSE-infected and three uninfected bovine brains were investigatedand used to demonstrate that the pentosan polysulphate, under thespecific conditions described, could specifically capture rogue prionprotein from the brain homogenates.

Method

Preparation of Pentosan Polysulphate Coated Magnetic Beads.

-   -   1. 600 μl of streptavidin superparamagnetic beads (Sigma-Aldrich        Company Ltd., S-2415) were washed by magnetic capture in three        consecutive 1 ml volumes of TBS (50 mM Tris, 150 mM NaCl, pH        7.5).    -   2. The beads were finally resuspended in 540 μl of TBS and 60 μl        of 10 mg/ml biotinylated pentosan polysulphate in TBS added. The        beads were incubated at room temperature for 1 hour with gentle        rocking to allow the pentosan polysulphate to coat the beads.    -   3. After coating the beads were washed by magnetic capture in        three consecutive 1 ml volumes of 5% (w/v) bovine albumin        (Sigma-Aldrich Company Ltd., A-7906), 50 mM phosphate buffer pH        8.4 and finally resuspended in 60 μl of the same buffer. The        beads were then ready for use.        Preparation of the Brain Homogenates.    -   1. 300-500 mg of each brain tissue was added to the grinding        tubes containing grinding beads as supplied in the BSE        Purification Kit (Bio-Rad). The liquid originally supplied in        these tubes in the kit was aspirated and discarded prior to use.    -   2. A volume of 150 mM NaCl that was calculated to generate a 50%        (w/v) brain homogenate after homogenisation was added to each        tube.    -   3. The tubes were homogenised for 45 seconds at speed setting        6.5 on a ribolyzer (purchased from Bio-Rad).    -   4. The homogenates were diluted 5-fold with 5% (w/v) bovine        albumin, 50 mM phosphate buffer pH 8.4.    -   5. 45 μl volumes of each homogenate were placed in separate        tubes.    -   6. 5 μl of 20% (w/v) SDS (sodium dodecyl sulfate) (Sigma-Aldrich        Company Ltd., L-5750) was added to each tube and mixed        thoroughly.    -   7. 450 μl of 5% (w/v) bovine albumin, 50 mM phosphate buffer pH        8.4 was then added to each and mixed.    -   8. 50 μl of 20% (w/v) N-lauroylsarcosine (Sigma-Aldrich Company        Ltd., L-9150) was then added and mixed.        Specific Capture of the Rogue Prion Protein    -   1. 10 μl of prepared pentosan polysulphate-coated        superparamagnetic beads were added to each diluted brain        homogenate and incubated with rocking for 1 hour at room        temperature.    -   2. Each reaction was then washed by magnetic capture with 3×100        μl volumes of TBS.        Elution of the Rogue Prion Protein and Immunodetection.    -   1. The beads from each reaction were resuspended in 10 μl of C1        (supplied with the Bio-Rad Platelia™ BSE Detection kit).    -   2. 5 μl of 0.2% (w/v) SDS was added to each bead suspension and        mixed.    -   3. 5 μl of 1M guanidine thiocyanate (Sigma-Aldrich Company Ltd.,        G-9277) was added to each bead suspension and mixed.    -   4. The reaction was heated at 100° C. for 5 minutes.    -   5. 100 μl of R6 (supplied with the Bio-Rad Platelia™ BSE        Detection kit) was then added and mixed.    -   6. 100 μl of each eluate was then used in the Bio-Rad.    -   7. Platelia™ BSE Detection kit using the protocol and reagents        supplied with this kit. Briefly, this kit involves immunocapture        of normal and/or rogue prion protein and immunodetection with a        horseradish peroxidase conjugated antibody.        Results

After performing the immunodetection in the microtiter plate-basedPlatelia™ assay the signal in each well was measured at a wavelength of450 nm using an ELISA reader. Brain homogenate used OD₄₅₀ BSE-infectedbovine 0.465 brain sample 1 BSE-infected bovine 0.382 brain sample 2BSE-infected bovine 0.437 brain sample 3 Normal bovine brain 0.060sample 1 Normal bovine brain 0.074 sample 2 Normal bovine brain 0.066sample 3

The signals from the three BSE-infected brain homogenates containingrogue prion protein is significantly higher than in the uninfectednormal brain homogenates.

Discussion

The Bio-Rad Platelia™ BSE Detection kit cannot differentiate betweennormal or rogue prion protein. Normally, the specificity for rogue prionprotein is achieved by prior digestion of the sample with proteinase Kwhich removes the protease susceptible normal prion protein. Any rogueprion protein in the sample is more resistant to protease digestion andremains and is subsequently detected by the Platelia™ assay. In thisexperiment we have demonstrated an alternative approach to proteasedigestion of the sample. We have used defined conditions under whichpentosan polysulphate can specifically capture the rogue prion proteinfrom the sample. This captured rogue prion protein is eluted anddetected in the immunoassay. Normal prion protein is not captured by thepentosan polysulphate and is washed away and is therefore not detectedin the immunoassay. We have demonstrated that by using this technique wecould correctly detect rogue prion protein in three BSE-infected bovinebrains and no signal was observed in three normal bovine brains.

EXAMPLE 3 Biotinylation of PPS Principle of the Method

Approximately one in ten of the sugar residues in the poly-xylosebackbone of pentosan sulphate is substituted with a uronic acid residue,this in turn is substituted with a methyl ester on some of the carboxylgroups, thus a number of free carboxyl groups exist in the molecule andcan be derivatised with carbodiimide to form active esters. These inturn may be substituted with amino species to generate an amide bond. Inthis particular case, EDC and NHS are chosen to form the active esterand biotin hydrazide is chosen as the amino species. Two reactions wereperformed, a one step reaction in which biotin hydrazide is presentinitially and no NHS is added, and a second reaction in which NHS/EDC isallowed to react simultaneously with PS and biotin hydrazide.

Materials

-   -   Pentosan sulphate (Norton Healthcare) was a gift from Stephen        Dealler    -   Biotin hydrazide 100 mg, Pierce#21339 mw 258.33 batch AH41461    -   EDC [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide methiodide]        Sigma #16,534-4, 1 g,    -   NHS[N-Hydroxysuccinimide] Sigma #H7377 5 g mw 115.1    -   Dialysis tubing mwco 3.5k Pierce # 68035    -   DMSO Sigma        Method

The two reactions were conducted using the following protocols in twoversions, with and without NHS.:

Dissolve 100 mg of biotin hydrazide in 6 ml of DMSO in a glass vial,this may require warming and/or ultrasonication. The final concentrationis thus 16.7 mg/ml or 65 mM. Take 1,000 mg of pentosan sulphate anddissolve in 10 ml of a 50/50 mixture of DMSO and water, this can be donein a plastic universal container. Dissolve 100 mg of EDC in 1 ml of DMSOin a glass vial, it may need warming. Dissolve NHS (approx 40-50 mg) in1.0 ml of water.

The reaction is performed in conical bottom polystyrene universalcontainers, with a small circular magnetic stirrer bar (approx 10 mmdia) on a magnetic stirrer base and fitted with a combination pHelectrode of 12 mm dia (or less).

EXAMPLE 3a Reaction Without NHS

Place 5.0 ml of pentosan sulphate solution in the reaction vessel, add1.0 ml of biotin hydrazide solution, stir well and record the pH. Avalue of 7-8 can be expected. Add 0.2 ml of EDC solution and whilstcontinuously stirring, record the pH and add 10 uL aliquots of 1 N HClfrom a glass micro-syringe and needle, recording the pH after everyaddition. Continue additions of acid until the pH is in the range 5-6.This is necessary as the reaction generates OH ions. The reaction shouldremain clear and colourless throughout. If any white precipitate ofbiotin hydrazide is formed, then the concentration of DMSO should beincreased, the target value is >/=50%. Leave the reaction for 2-3 hoursat room temperature (or overnight if this is more convenient).

Record the final pH of the reaction mixture. Add an equal volume of 1MNaCl to dilute the DMSO down to 25% and displace tonically boundhydrazide and transfer the entire contents to a 35 cm length of 2.2 cmdia dialysis tubing. Note the DMSO concentration is reduced to 25% toavoid damage to the dialysis tubing, the tubing should also be testedwith water prior to use to detect any pinholes and should be only ⅓ fullto allow for swelling on dialysis. Dialyse overnight against 2L of waterand repeat this several times, the more dialysis the better as pentosansulphate tends to strongly retain basic ions by non-covalent ionicinteraction by virtue of its strong negative charge. Freeze dry thedialysed solution and record the dry weight. The final product should bea firm white cake. Yields can vary a lot, but 50-60% is typical, most ofthe loss occurs on dialysis, due to MW heterogeneity of the pentosansulphate and loss of species with a MW of less than 3,500.

EXAMPLE 3b Reaction with NHS

This reaction is carried out essentially as above except that 1.0 ml (44mg) of NHS is added to the reaction vial prior to the addition of theEDC reagent which starts the reaction. The initial pH may be in therange of 6-7 and should be adjusted down with 1 N HCl to approx pH 5-6.

Quality Control

After calculating the recovery from the dry weight, make up a solutionof 10 mg/ml in water and scan the spectrum from 200 to 400 nm. Peaksshould be seen at 260 and 280 nm, though one or both may be unresolvedshoulders. This adsorption is due to pyridine residues incorporated intothe molecule during the sulphation step. They can be used to monitor theconcentration of pentosan sulphate, eg during chromatography. Pentosancan be monitored by UV absorption at 260 nm, or at lower concentrationsby the Toluidine Blue metachromasia assay.

EXAMPLE 4 Removal of Prion Protein from Plasma

Removal of the Rogue Prion Protein

-   -   1. 100 μl of prepared pentosan polysulphate-coated        superparamagnetic beads were added to one of two PrP^(Sc) spiked        freshly prepared human plasma aliquots. Both aliquots were        incubated with rocking for 1 hour at room temperature.    -   2. The beads were then removed from the spiked plasma aliquot by        magnetic capture. This supernatant, together with the remaining        plasma aliquot were then tested for the presence of the rogue        prion protein.        Testing of the Spiked Aliquots for Rogue Prion Protein.    -   1. The two plasma aliquots were treated with proteinase K under        conditions that we have shown to digest normal prion protein but        leave rogue protein intact. These conditions are easily        determined empirically. The proteinase K treated samples were        then tested for the presence of the rogue prion protein using        the immunobased Bio-Rad Platelia™ BSE Detection kit.        Results

After performing the immunodetection in the microwell plate-basedPlatelia™ assay the signal in each well was measured at a wavelength of450 nm using an ELISA reader. The rogue prion protein could be readilydetected in the spiked serum sample that had not been treated withpentosan polysulphate. In contrast the pentosan polysulphate-treatedsample gave no signal in the test demonstating that there was nodetectable rouge prion protein remaining in this sample.

Discussion

This experiment demonstrates that pentosan polysulphate can be used toeffectively remove rogue prion protein from samples of interest.

EXAMPLE 5 Investigation of Detergent Conditions Allowing the SpecificBinding of Pentosan Polysulphate to the Rogue Prion Protein

Introduction

Biotin was conjugated to pentosan polysulphate using standard chemicalmethods. The biotinylated pentosan polysulphate was used to coatstreptavidin-derivatised superparamagnetic beads. The coated beads werethen used to establish conditions of detergent under which the pentosanpolysulphate could bind the rogue prion protein but not the normalcellular prion protein.

Method

Preparation of Pentosan Polysulphate Coated Magnetic Beads.

-   -   1. 600 μl of streptavidin superparamagnetic beads (Sigma-Aldrich        Company Ltd., S-2415) were washed by magnetic capture in three        consecutive 1 ml volumes of TBS (50 mM Tris, 150 mM NaCl, pH        7.5).    -   2. The beads were finally resuspended in 540 μl of TBS, 5% (w/v)        bovine albumin (BSA) (Sigma-Aldrich Company Ltd., A-7906) and 60        μl of 10 mg/ml biotinylated pentosan polysulphate in TBS added.        The beads were incubated at room temperature for 1 hour with        gentle rocking to allow the pentosan polysulphate to coat the        beads.    -   3. After coating the beads were washed by magnetic capture in        three consecutive 1 ml volumes of, 50 mM phosphate buffer pH        8.4, 5% (w/v) BSA and finally resuspended in 60 μl of the same        buffer. The beads were then ready for use.        Preparation of the Brain Homogenates.    -   1. 300-500 mg samples of BSE-infected and normal bovine brain        tissue were added to the grinding tubes containing grinding        beads as supplied in the BSE Purification Kit (Bio-Rad). The        liquid originally supplied in these tubes in the kit was        aspirated and discarded prior to use.    -   2. A volume of 150 mM NaCl that was calculated to generate a 50%        (w/v) brain homogenate after homogenisation was added to each        tube.    -   3. The tubes were homogenised for 45 seconds at speed setting        6.5 on a ribolyzer (purchased from Bio-Rad).    -   4. The homogenates were diluted 5-fold with 5% (w/v) BSA, 50 mM        phosphate buffer pH 8.4.    -   5. 45 μl volume aliquots of each homogenate were placed in        separate tubes.    -   6. 5 μl of 20% (w/v) SDS (sodium dodecyl sulfate) (Sigma-Aldrich        Company Ltd., L-5750) was added to each tube and mixed        thoroughly.    -   7. 450 μl of 5% (w/v) BSA, 50 mM phosphate buffer pH 8.4 was        then added to each aliquot and mixed.    -   8. 50 μl of N-lauroylsarcosine (Sigma-Aldrich Company Ltd.,        L-9150) at various concentrations of detergent was then added to        various aliquots and mixed. One set (one BSE-infected and one        uninfected brain) had no N-lauroylsarcosine added.        Capture of Prion Protein    -   1. 10 μl of prepared pentosan polysulphate-coated        super-paramagnetic beads were added to each diluted brain        homogenate and incubated with rocking for 1 hour at room        temperature.    -   2. Each reaction was then washed by magnetic capture with 3×100        μl volumes of TBS.        Elution of the Prion Protein and Immunodetection    -   1. The beads from each reaction were resuspended in 10 μl of C1        (supplied with the Bio-Rad Platelia™ BSE Detection kit).    -   2. 5 μl of 0.2% (w/v) SDS was added to each bead suspension and        mixed.    -   3. 5 μl of 1M guanidine thiocyanate (Sigma-Aldrich Company Ltd.,        G-9277) was added to each bead suspension and mixed.    -   4. The reaction was heated at 100° C. for 5 minutes.    -   5. 10 μl of R6 (supplied with the Bio-Rad Platelia™ BSE        Detection kit) was then added and mixed.    -   6. 10 μl of each eluate was then used in the Bio-Rad Platelia™        BSE Detection kit using the protocol and reagents supplied with        this kit. Briefly, this kit involves immunocapture of normal        and/or rogue prion protein and immunodetection with a        horseradish peroxidase conjugated antibody.        Results

After performing the immunodetection in the microtiter plate-basedPlatelia™ assay the signal in each well was measured at a wavelength of450 nm using an ELISA reader. Final concentration of N-lauroylsarcosinein the bead capture buffer Bovine brain used OD₄₅₀   2% BSE-infectedbrain 0.52   2% Normal brain 0.14   1% BSE-infected brain 0.33   1%Normal brain 0.13 0.5% BSE-infected brain 0.45 0.5% Normal brain 0.130.2% BSE-infected brain 0.41 0.2% Normal brain 0.09   0% BSE-infectedbrain 0.24   0% Normal brain 0.86

At all concentrations of N-lauroylsarcosine there was a discriminationbetween BSE-infected and normal brain. 0.2% N-lauroylsarcosine was thebest concentration of detergent and allowed the pentosan polysulphate tobind to and capture the rogue prion protein without binding or captureof the normal prion protein. In the absence of N-lauroylsarcosine, eventhough SDS detergent was present, there was no discrimination ofpentosan polysulphate binding to rogue prion and normal prion protein.Under these conditions the pentosan polysulphate bound both the normaland rogue prion protein.

Discussion

-   -   1. The specificity of binding of pentosan polysulphate to rogue        prion protein under these specific test conditions is dependent        upon the presence of N-lauroylsarcosine or similar detergents.        Without this detergent the pentosan polysulphate bound to both        normal and rogue prion protein.

EXAMPLE 6 Investigation of pH Conditions Allowing the Specific Bindingof Pentosan Polysulphate to the Rogue Prion Protein

Introduction

Biotin was conjugated to pentosan polysulphate using standard chemicalmethods. The biotinylated pentosan polysulphate was used to coatstreptavidin-derivatised super-paramagnetic beads. The coated beads werethen used to establish conditions of pH under which the pentosanpolysulphate could bind the rogue prion protein but not the normalcellular prion protein.

Method

Preparation of Pentosan Polysulphate Coated Magnetic Beads.

-   -   1. 1 ml aliquots of streptavidin superparamagnetic beads        (Sigma-Aldrich Company Ltd., S-2415) were washed by magnetic        capture in three consecutive 1 ml volumes of TBS (50 mM Tris,        150 mM NaCl, pH 7.5).    -   2. Each aliquot of beads were finally resuspended in 1 ml of TBS        5% (w/v) bovine serum albumin (BSA) (Sigma-Aldrich Company Ltd.,        A-7906) and 100 μl of 10 mg/ml biotinylated pentosan        polysulphate in TBS added. The beads were incubated at room        temperature for 1 hour with gentle rocking to allow the pentosan        polysulphate to coat the beads.    -   3. After coating, each aliquot of beads was washed by magnetic        capture in three consecutive 1 ml volumes of 5% (w/v) BSA, 50 mM        Tris buffer pH 8.4.    -   4. Aliquots of beads were then resuspended in buffers of pH 5.7,        7.5, 8.4 and 9.6 all containing 5% (w/v) BSA.        Preparation of the Brain Homogenates in Buffers of Various pH    -   1. 300-500 mg of BSE-infected and normal bovine brain tissue        were each added to a grinding tube containing grinding beads as        supplied in the BSE Purification Kit (Bio-Rad). The liquid        originally supplied in these tubes in the kit was aspirated and        discarded prior to use.    -   2. A volume of 150 mM NaCl that was calculated to generate a 50%        (w/v) brain homogenate after homogenisation was added to each        tube.    -   3. The tubes were homogenised for 45 seconds at speed setting        6.5 on a ribolyzer (purchased from Bio-Rad).    -   4. 50 μl of each homogenate was diluted 5-fold in buffers of pH        5.7, 7.5, 8.4 and 9.6 all containing 5% (w/v) BSA.    -   5. 45 μl volumes of each diluted homogenate were placed in        separate tubes.    -   6. 5 μl of 20% (w/v) SDS (sodium dodecyl sulfate) (Sigma-Aldrich        Company Ltd., L-5750) was added to each tube and mixed        thoroughly.    -   7. 450 μl of buffer of the same pH as the initial dilution        buffer all containing 5% (w/v) bovine albumin was then added to        each and mixed.    -   8. 50 μl of 20% (w/v) N-lauroylsarcosine (Sigma-Aldrich Company        Ltd., L-9150) was then added and mixed.        Bead Capture of the Brain Homogenates    -   1. 10 μl of prepared pentosan polysulphate-coated        super-paramagnetic beads in buffer of the corresponding pH were        added to each diluted brain homogenate and incubated with        rocking for 1 hour at room temperature.    -   2. Each reaction was then washed by magnetic capture with 3×100        μl volumes of TBS.        Elution of the Rogue Prion Protein and Immunodetection.    -   1. The beads from each reaction were resuspended in 10 μl of C1        (supplied with the Bio-Rad Platelia™ BSE Detection kit).    -   2. 5 μl of 0.2% (w/v) SDS was added to each bead suspension and        mixed.    -   3. 5 μl of 1M guanidine thiocyanate (Sigma-Aldrich Company Ltd.,        G-9277) was added to each bead suspension and mixed.    -   4. The reaction was heated at 100° C. for 5 minutes.    -   5. 100 μl of R6 (supplied with the Bio-Rad Platelia™ BSE        Detection kit) was then added and mixed.    -   6. 100 μl of each eluate was then used in the Bio-Rad Platelia™        BSE Detection kit using the protocol and reagents supplied with        this kit. Briefly, this kit involves immunocapture of normal        and/or rogue prion protein and immunodetection with a        horseradish peroxidase conjugated antibody.        Results

After performing the immunodetection in the microtiter plate-basedPlatelia™ assay the signal in each well was measured at a wavelength of450 nm using an ELISA reader. pH buffer used Bovine brain used OD₄₅₀ 5.7BSE-infected brain 0.79 5.7 Normal brain 0.30 7.5 BSE-infected brain1.57 7.5 Normal brain 1.25 8.4 BSE-infected brain 0.42 8.4 Normal brain0.04 9.6 BSE-infected brain 0.08 9.6 Normal brain 0.04

At a pH of 7.5 and lower the pentosan polysulphate-coated beads couldbind both normal and rogue prion protein. At pHs of 9.6 and higher thepentosan polysulphate-coated beads could not bind both forms of theprion protein. At pH 8.4 the pentosan polysulphate-coated beads capturedthe rogue prion protein but did not capture the normal prion protein. Atthis pH the pentosan polysulphate shows specificity for binding to therogue prion protein.

Discussion

The specificity of binding under the test conditions of pentosanpolysulphate to rogue prion protein is dependent upon the pH. At pH 8.4pentosan polysulphate binds rogue prion protein but cannot bind thenormal prion protein. At pHs of 7.5 and lower both normal and rogueprion are bound whereas at pHs of 9.6 and higher there is no binding ofrogue or normal prion protein. Therefore, for specific binding ofpentosan polysulphate to rogue prion protein under these conditions a pHclose to 8.4 should be used.

EXAMPLE 7 Demonstration of Specific Capture of PrP^(res) (PrP^(Sc)) to aHigh Charge Density Polyanionic Ligand Using Competing Lower ChargeDensity Polyanions to Selectively Inhibit Binding of PrP^(c)

Background

PrP can be bound to immobilised polyanions. In the absence of competingpolyanions in the capture buffer both PrP^(res) and PrP^(c) arecaptured. Specificity for capture of PrP^(res) can be achieved byincluding in the capture buffer a polyanion of lower charge density thanthat of the capture polyanion. In this example dextran sulphate is usedas the high charge density capture polyanion and N-lauroyl sarcosine(which forms multi-molecular detergent micelles) and pentosanpolysulphate or fucoidan are used as the weaker charge density competingpolyanions.

Method

-   -   1. Maxisorp microtitre wells were coated with dextran sulphate        (500 000 mwt) following standard procedures.    -   2. 100 μl of brain homogenate containing 1 mg brain, 50 mM Tris        pH 8.3, 1% (w/v) BSA, 1% (v/v) Triton X-100 were added to the        coated wells. In some cases this capture buffer also contained        either 1% (w/v) N-lauroyl sarcosine, fucoidan, dextran sulphate        or various concentrations of pentosan polysulphate.    -   3. After incubation for 2 hours to allow capture of prion        protein, the wells were washed ×3 with 50 mM Tris pH 8.3, 1%        (w/v) BSA, 1% (v/v) Triton X-100.    -   4. The wells were then washed ×3 with PBS.    -   5. 100 μl of 5M guanidinium thiocyanate was added to each well        and incubated 5 mins at 4° C.    -   6. Wells were washed 3× with PBS and then captured prion protein        detected with the anti-prion protein conjugate from the Bio-rad        Platelia™ BSE-detection kit following the kit protocol.    -   7. Developed signal was measured in an ELISA reader at OD450.

Results BSE-infected or normal Competing polyanion used brain OD450 NoneBSE-infected 0.10 None Normal 0.15 1% (w/v) N-lauroyl BSE-infected 0.95sarcosine 1% (w/v) N-lauroyl Normal 0.03 sarcosine 1 mg/ml pentosanBSE-infected 0.26 polysulphate 1 mg/ml pentosan Normal 0.03 polysulphate0.1 mg/ml pentosan BSE-infected 0.14 polysulphate 0.1 mg/ml pentosanNormal 0.07 polysulphate 1 mg/ml fucoidan BSE-infected 0.13 1 mg/mlfucoidan Normal 0.03 1 mg/ml dextran sulphate BSE-infected 0.02 1 mg/mldextran sulphate Normal 0.03Discussion

In the absence of competing polyanion in the capture buffer the overallsignal is lower and there is no difference in signal from infected ornormal brain i.e. there is no specific capture of PrP^(res). The signalfrom infected brain, however, is increased by including a competingpolyanion in the capture buffer and the signal from the correspondingnormal or uninfected brain is suppressed. In this example, the bestdifferentiation between infected and normal brain is achieved by the useof 1% (w/v) N-lauroyl sarcosine in the capture buffer. In addition, adifferentiation between infected and normal brain can be achieved withfucoidan or pentosan polysulphate. With pentosan polysulphate thedifferentiation can be increased by increasing the concentration of thecompeting polyanion, pentosan polysulphate, in the capture buffer from0.1 to 1 mg/ml. As a control, if dextran sulphate is included in thecapture buffer the signal, as expected, is reduced to background as itcompetes for and inhibits the binding of the PrP to the immobiliseddextran sulphate.

EXAMPLE 8 Demonstration of Specific Capture of PrP^(res) to a HighCharge Density Polyanion Coated Surface

Background

In this experiment it was demonstrated that PrP^(res) could bespecifically captured to a polyanionic surface. In this instance, thesurface was provided by derivatised maleic anhydride polystyrene.Uncharged polysorp and maxisorp wells were used as controls. In otherexperiments it has been demonstrated that these uncharged surfaces canbe derivatised with polyanionic dextran sulphate and can then bindPrP^(res).

Method

-   -   1. Maleic anhydride activated polystyrene microplate wells        (Perbio Science UK Ltd., Cheshire) were derivatised with TBS 5%        (w/v) BSA for 60 mins at room temperature. This generates a        carboxyl charged surface on the plastic (see product        literature). As non-charged controls, polysorp and maxisorp        wells (Nunc) were also investigated. In addition, maxisorp wells        were also coated with a polyanionic dextran sulphate ligand        using the procedure described in Example 9.    -   2. 100 μl of brain homogenates containing 1 mg infected or        uninfected brain in 50 mM Tris pH 8.3, 1% (w/v) BSA, 1% (v/v)        Triton X-100, 1% (w/v) N-lauroyl sarcosine were added to the        wells.    -   3. After incubation for 2 hours to allow capture of prions, the        wells were washed ×3 with 50 mM Tris pH 8.3, 1% (w/v) N-lauroyl        sarcosine.    -   4. The wells were then washed ×3 with PBS. 5. 100 μl of 5M        guanidinium thiocyanate was added to each well and incubated 5        mins at 4° C.    -   6. Wells were washed 3× with PBS and then captured prion        detected with the anti-prion conjugate from the Bio-Rad        Platelia™ BSE-detection kit following the kit protocol.    -   7. Developed signal was measured in an ELISA reader at OD450.

Results Type of wells used BSE-infected or normal brain OD450 AnionicBSE-infected 0.2 Anionic Normal 0.03 Polysorp BSE-infected 0.05 PolysorpNormal 0.03 Maxisorp BSE-infected 0.02 Maxisorp Normal 0.02 Maxisorpcoated with BSE-infected 1.0 dextran sulphate Maxisorp coated withNormal 0.02 dextran sulphateDiscussion

The anionic polystyrene surface, under the conditions used in thisexperiment, specifically captured PrP^(res). Uncharged plastic did nothave this effect unless it had been coated with a polyanionic ligand.

EXAMPLE 9 Study of Effects of Dilution of Positive Brain Sample inNegative Sample

Material

-   -   Positive Sample: A 25% suspension of brain homogenate known to        be positive for PrP^(sc)    -   Negative Sample: A 25% suspension of brain homogenate known to        be negative for PrP^(sc)        Preparation

Maxisorb plates were coated according to the following coating protocol.1 mg of Polybrene was coated onto the plates in carbonate buffer at pH7.4 and left overnight, washed 3 times with PBS. The plates were thencoated with 1 mg of dextran sulphate in PBS. After 6 hours, the plateswere washed 3 times with PBS, then blocked with 5% BSA by adding 400 μlof 5% BSA solution and leaving for 30 minutes. Plates were then washed 3times with PBS and allowed to dry.

Sample Preparation

Preparation of Sample Dilution in Negative Brain sample Method Neat +ve40 μl of +ve sample 1/5  8 μl of +ve sample + 32 μl of −ve sample 1/10 5 μl of +ve sample + 45 μl of −ve sample 1/100  6 μl of (1/10 diluted+ve sample) + 54 μl of −ve sample 1/250 20 μl of (1/100 diluted +vesample) + 30 μl of −ve sample 1/1000 10 μl of (1/250 diluted +vesample) + 30 μl of −ve sample Neat −ve 25 μl of −ve sample

Preparation of Sample Dilution in Water sample Method Neat +ve 40 μl of+ve sample 1/5  8 μl of +ve sample + 32 μl of H₂O 1/10  5 μl of +vesample + 45 μl of H₂O 1/100  6 μl of (1/10 diluted +ve sample) + 54 μlof H₂O 1/250 20 μl of (1/100 diluted +ve sample) + 30 μl of H₂O 1/100010 μl of (1/250 diluted +ve sample) + 30 μl of H₂OSample Preparation Prior to Running in Assay

40 μl of sample was mixed with 60 μl of H₂O and 25 μl of capture buffer,250 mM Tris pH 8.4, 5% BSA, 5% Triton X-100, 5% sarkosyl, 1.25 mg/mltrypsin.

Assays were performed according to the following Assay Protocol:

-   -   1. Add 100 μl of sample to plate and incubate at RT for 120        minutes.    -   2. Wash ×3 with 50 mM Tris pH8.4 +1% sarkosyl and ×3 with PBS.    -   3. Add 10 μl of 4MGuSCN in 20% PEG and incubate for 10 minutes        at 2-8° C.    -   4. Wash ×3 with PBS.    -   5. Add 100 μl of Bio-Rad Platelia™ enzyme antibody conjugate and        incubate at 2-8° C. for 60 minutes.    -   6. Wash ×5 with Bio-Rad Platelia™ wash.    -   7. Add 100 μl of Bio-Rad Platelia™ substrate and incubate for 30        minutes in dark.    -   8. Add 10 μl of Bio-Rad Platelia™ stop solution and read.

Plate Layout 1 1 A Neat +ve 1/10 In H₂O B 1/5 In negative 1/100 In H₂Obrain C 1/10 in negative 1/250 In H₂O brain D 1/100 In negative 1/1000In H₂O brain E 1/250 in negative brain F 1/1000 in negative brain G Neat−ve H 1/5 In H₂OThe results obtained were as follows:

-   -   Dilution of 10 mg of brain

homogenate in −ve brain sample mg of −ve mg of +ve Dilution label brainbrain Factor OD Neat +ve 0.00 10.00 1 4 1/5 8.00 2.00 5 1.992 1/10 9.001.00 10 1.252 1/100 9.90 0.10 100 0.175 1/250 9.96 0.04 250 0.077 1/10009.99 0.01 1000 0.039 Neat −ve 10.00 0.00 0 0.021

-   -   Dilution of 10 mg of brain

homogenate in H₂O sample mg of −ve mg of +ve Dilution label brain brainFactor OD Neat +ve 0.00 10.00 1 4 ⅕ 0.00 2.00 5 2.377 1/10 0.00 1.00 101.395 1/100 0.00 0.10 100 0.145 1/250 0.00 0.04 250 0.053 1/1000 0.000.01 1000 0.016

Summary OD mg of +ve Diluted in Diluted in brain −ve brain H2O 10.00 4 42.00 1.992 2.377 1.00 1.252 1.395 0.10 0.175 0.145 0.04 0.077 0.053 0.010.039 0.016 0.00 0.021 —

These results are presented graphically in FIG. 1, which shows thedilution curves for dilution of positive brain with respectively waterand negative brain. The two curves are essentially the same,demonstrating that the presence of negative brain material does notinterfere with the assay.

EXAMPLE 10 Capture of Aggregated Tau Protein in Alzheimer's Brain andNormal Age-Matched Controls

We have shown that, under defined conditions, various selective captureagents are specific for the capture of aggregated pathogenic prionprotein such that normal unaggregated prion is not captured. Theaggregated prion protein has an extensive beta-pleated sheet structurewhereas normal prion is mostly alpha helix in structure. This exampledemonstrates that other aggregated beta-pleated sheet proteins such astau aggregates that are found in Alzheimer's Disease can similarly beselectively captured.

Method

-   -   1. 25% (w/v) homogenates of Alzheimer's and age matched control        brains were prepared in distilled water.    -   2. 4 μl of brain was made up to 100 μl in Capture buffer (50 mM        Tris pH 8.4, 1% (v/v) Triton X-100, 1% (w/v) N-lauroyl        sarcosine, 1% (w/v) BSA).    -   3.25 μl of brain was also made up to 100 μl in Capture buffer        containing 25 μg Trypsin.    -   4. Duplicate 100 μl aliquots of brain prepared as in steps 2 and        3 above were added to dextran sulphate-coated microtiter wells        and incubated for 2 hours at room temperature.    -   5. Wells were then washed three times with 50 mM Tris pH 8.4, 1%        (w/v) N-lauroyl sarcosine.    -   6. Samples were incubated with an anti-tau monoclonal antibody        in PBS 0.1% (v/v) Tween20.    -   7. After 1 hour at room temperature wells were washed three        times with PBS 0.1% (v/v) Tween20.    -   8. Immobilised primary antibody was detected with an anti-mouse        IgG horseradish peroxidase conjugate following standard        procedures.        Results

Results with the Anti-Tau Antibody 1 mg brain 10 mg brain Brain Notrypsin With trypsin Alzheimer's 1 1.32 1.26 Alzheimer's 2 0.85 0.62Control 1 0.56 0.20 Control 2 0.97 0.51Discussion

It is known that the brains from most aged individuals containaggregated tau but in Alzheimer's Disease there are more of theseaggregates than in age matched controls. Here, the selective captureagent is capturing these aggregates. In this example, trypsin digestiondecreases the binding of the protein and reduces the signal but, underthese conditions, does not reduce it to back-ground. The ratio of signalafter treatment with trypsin to the signal without treatment was muchhigher in the Alzheimer's brains than in the controls. This suggeststhat there is more protease resistant aggregates of tau protein inAlzheimer's brain compared to the age matched controls.

EXAMPLE 11 The Effect of Titrating Trypsin on PrP^(Sc) Positive Samples

Method

Dextran Sulphate Coated Plate:

1 mg of Hexadimethrine bromide (Polybrene) (100 μl of 10 mg/ml incarbonate buffer pH 7.4) was coated onto Maxisorb plates and left overnight at RT°.

Each plate was then washed 3 times with PBS and coated with 1 mg ofDextran Sulphate (MW 500000) (10 mg/ml stock in Tris buffer pH 8.6) andleft at RT° for 4 hrs.

The plates were then washed 3 times with PBS and then blocked with 300μl of 5% BSA solution in TBS and left at RT° for 30 minutes.

The plates were then washed 3 times with PBS.

Capture Buffer

250 mM Tris buffer at pH 8.4 containing 5% BSA, 5% Sarkosyl, 5% Triton

Sample

Weakly and strongly positive brains BI63 and SV10 (25% homogenate) weretreated as follows to provide samples for assay.

25 μl of brain homogenate +25 μl of capture buffer, 250 mM Tris pH 8.4,5% BSA, 5% Triton X-100, 5% sarkosyl, +65 μl of H₂O.

To this sample 10 μl of various concentrations of Trypsin was added.

Wash Buffer

50 mM Tris pH8.4 +1% sarkosyl

Method

Assay Protocol

-   -   1. Add 100 μl of sample to plate and incubate at RT for 120        minutes.    -   2. Wash ×3 with 50 mM Tris pH8.4 +1% sarkosyl and ×3 with PBS.    -   3. Add 100 μl of 4MGuSCN (in 20% PEG) and incubate for 10        minutes at 2-8° C.    -   4. Wash ×3 with PBS.    -   5. Add 100 μl of Bio-Rad Platelia™ enzyme antibody conjugate and        incubate at 2-8° C. for 60 minutes.    -   6. Wash ×5 with Bio-Rad Platelia™ wash.    -   7. Add 100 μl of Bio-Rad Platelia™ substrate and incubate for 30        minutes in dark.    -   8. Add 100 μl of Bio-Rad Platelia™ stop solution and read.        Results

-   5 mg of Positive

brain Bi63 Trypsin (μg) OD 1000 0.135 100 0.14 25 0.169 10 0.173 1 0.0680 0.068

-   5 mg Positive

brain Sv10 Trypsin (μg) OD 25 2.858 0 0.894Conclusion

The presence of Trypsin appears to have increased the signal. It alsoappears that a broad concentration range of Trypsin can be used withouta detrimental effect on assay.

EXAMPLE 12 Demonstration of the Specific Binding of Prp^(SC) by PolyCations

Method

This example demonstrates the use of various poly cations for specificcapture of PrP^(Sc). The ligands were either passively coated ontopolystyrene microplates or actively coated (i.e. bound), whereappropriate, to maleic anhydride plates.

Selective Binding Agent Immobilisation

All the selective binding agents were immobilised overnight at 16-25° C.in 50 mM carbonate buffer pH 9.6 at a concentration of 10 μg/ml. Afterimmobilization, the wells were washed ×3 with PBS and then blocked with5% (w/v) BSA in PBS for 30 mins. After blocking, wells were washed ×2with PBS before use. The PAMA dendrimer starbust, poly L-lysine andpolyethyleneimine were coated onto both Maxisorp and maleic anhydridemicroplates whereas the polybreen and pDADMAC were coated onto theMaxisorp plates only.

Capture of PrP^(SC)

-   -   1. BSE-infected bovine and uninfected bovine brains were        homogenized in distilled water following commercially defined        protocols.    -   2. 0.5 mg of homogenised brain was captured in ligand coated        wells in a total volume of 100 μl 50 mM Tris pH 8.3, 1% (w/v)        N-lauroyl sarcosine, 1% (v/v) Triton X-100, 1% (w/v) BSA, 0.5        mg/ml trypsin (porcine pancreas).    -   3. After capture for 2 hours at 18-25° C. the wells were washed        ×3 with 50 mM Tris pH 8.3, 1% (w/v) N-lauroyl sarcqsine.    -   4. The wells were then washed ×3 with PBS and incubated for 10        mins with 100 μl of 4M guanidinium thiocyanate, 20% PEG 8000 at        4-8° C.    -   5. The wells were washed ×3 with PBS and then incubated with an        anti-prion monocolonal antibody horseradish peroxidase        conjugate.    -   6. After 60 mins the wells were washed ×5 with PBS 0.1% (v/v)        Tween20 and 100 μl TMB substrate added.    -   7. After 30 mins the OD450 of each reaction was measured and        recorded (see table below).

Results Passive Active adsorption adsorption Binding Agent PositiveNegative Positive Negative PAMA dendrimer 0.938 0.030 0.097 0.026starburst Polybreen 0.019 0.016 ND ND Poly L-lysine 0.070 0.017 0.0010.001 pDADMAC* 1.828 0.037 ND ND polyethyleneimine 0.118 0.030 0.4020.055*Aldrich 40903-0-mw 400,000-500,000Discussion

The pDADMAC and PAMA dendrimer starburst poly cations work well asPrP^(Sc)-specific ligands when passively coated to polystyrenemicroplates. The PDADMAC works best in this series of binding agents.Polyethyleneimine works to some degree when immobilised on maleicanhydride microplates through its amino groups.

This experiment demonstrates that a variety of poly cations can be usedto specifically capture PrP^(Sc) from infected brain under the givenCapture Buffer conditions used. These agents can be passively oractively immobilised to polystyrene surfaces. Other experiments havedemonstrated that maximum signal from 20 mg of positive brain can beachieved in the presence of 1% (w/v) N-lauroyl sarcosine in the CaptureBuffer; without N-lauroyl sarcosine the signal is reduced. Thisillustrates that the capture agents perform best under defined bufferconditions.

EXAMPLE 13 Capture of Aggregated Beta Amyloid and Tau in Alzheimer'sBrain and Normal Age-Matched Controls by Polycationic Binding Agent

Background

pDADMAC, under defined conditions, has been shown to be specific for thecapture of aggregated pathogenic prion protein; normal unaggregatedprion is not captured. The aggregated prion protein has an extensivebeta-pleated sheet structure whereas normal prion is mostly alpha helixin structure. It is postulated that the binding agent may recognizeother aggregated beta-pleated sheet proteins such as beta-amyloid andtau aggregates that are found in Alzheimer's disease. The experimentsbelow were performed in order to investigate this hypothesis.

Method

-   -   1.25% (w/v) homogenates of Alzheimer's and age matched control        brains were prepared in distilled water.    -   2. 80 μl of brain homogenate was made up to 100 μl in Capture        buffer (50 mM Tris pH 8.4, 1% (v/v) Triton X-100, 1% (w/v)        N-lauroyl sarcosine, 1% (w/v) BSA) and added to        polycationic-coated microwells (formed by coating the wells with        poly(diallyldimethyl ammonium chloride) (pDADMAC), (Aldrich        Chemical Company Inc., catalogue number 40,903-0).    -   3. After incubation for 2 hours at room temperature, the wells        were washed three times with 50 mM Tris pH 8.4, 1% (w/v)        N-lauroyl sarcosine and then incubated with an anti-tau        monoclonal antibody in PBS 0.1% (v/v) Tween20.    -   4. After 1 hour at room temperature wells were washed three        times with PBS 0.1 (v/v) Tween20.    -   5. Immobilised primary antibody was detected with an antimouse        IgG horseradish peroxidase conjugate following standard        procedures.

Results Classification Brain by brain bank OD450  67/97 Positive 1.85 73/97 Positive 0.80 163/97 Positive 0.61 149/97 Positive 0.45  97/97Negative 0.05  98/98 Negative 0.08Discussion

The polycationic binding agent enables capture of the tau aggregates.When the captured tau is detected with the anti-tau antibody, theAlzheimer's disease brains all gave a high positive signal whereas thenegative control brains gave a low negative signal. In conclusion,capture with a polycation under the specified conditions can enabledifferentiation of Alzheimer's disease brains from those brains withoutthe disease.

EXAMPLE 14 Effect of Different Proteases and DNase on the MatrixInhibition of pDADMAC Capture of PrP^(Sc)

Background

The effect of different proteases on the effectiveness of capture ofPrP^(Sc) to polycation-coated plates (formed by coating the wells withpoly(diallyldimethyl ammonium chloride) (pDADMAC), (Aldrich ChemicalCompany Inc., catalogue number 40,903-0) were investigated.

Method

-   -   1. 80 μl of brain homogenate was made up to 100 μl by addition        of 20 μl of Capture buffer (250 mM Tris pH 8.3, 5% (v/v) Triton        X-100, 5% (w/v) N-lauroyl sarcosine, 5% (w/v) BSA) containing        different proteases and/or DNase.    -   2. The homogenates were then added to polycationic-coated        microwells.    -   3. After incubation for 2 hours at room temperature, the wells        were washed six times with PBS.    -   4. 100 μl 4M Guanidine thiocyanate, 20% (w/v) PEG was added to        each well.    -   5. After incubation for 10 minutes at room temperature wells        were washed three times with PBS.    -   6. 100 μl of anti-prion protein horseradish peroxidase conjugate        (diluted 1:1500 in PBS 0.1% (v/v) Tween 20 and 5% (w/v) BSA) was        added.    -   7. After 1 hour at room temperature wells were washed five times        with PBS 0.1% (v/v) Tween20.    -   8. Immobilised conjugate was detected with TMB solution        following standard protocols.        Results

Assessing Effects of Chymotrypsin, Trypsin, DNase and Proteinase K inCapture Buffer Infected Protease or DNase used bovine brain No proteaseor DNase 0.122 Chymo/Trypsin (Conc both 0.139 6.25 mg/ml) Dnase/Trypsin(Conc 1 mg/ml 0.639 Dnase, 6.25 mg/ml Trypsin) Chymo/Dnase (Conc 1 mg/ml0.616 Dnase, 6.25 mg/ml Chymo) Chymo/Dnase/Trypsin Conc 0.460 1 mg/mlDnase, 6.25 mg/ml Chymo and Trypsin) Trypsin (Conc 6.25 mg/ml) 0.568Chymo (Conc 6.25 mg/ml) 0.171 Dnase (Conc 1 mg/ml) 0.180 Proteinase K(Conc 1 mg/ml) 0.531 Pronase (Conc 1.25 mg/ml) 0.222 Pronase (Conc 6.25mg/ml) 0.178

Titrating Trypsin and Chymotrypsin Concentrations in Capture BufferInfected Protease used bovine brain Trypsin 6.25 mg/ml 0.732 Trypsin1.25 mg/ml 0.726 Chymo 3.125 mg/ml 0.568 Chymo 0.625 mg/ml 0.433Discussion

It has been demonstrated that the polycationic ligand under certainconditions is specific for binding to PrP^(Sc). However, the signal canbe reduced by matrix effects derived from constituents of the brainhomogenate that can interfere with binding and reduce the signal. Thismatrix effect can be reduced and the signal from infected brainincreased by the use of proteases. This study shows that trypsin,chymotrypsin and proteinase K are effective at removing the matrixinhibition; pronase (at the concentrations investigated) is lesseffective. Trypsin at a concentration of 6.25-1.15 mg/ml is equallyeffective whereas chymotrypsin is more effective as the concentration isincreased. DNase has a demonstrable but smaller effect on removal ofmatrix inhibition.

EXAMPLE 15 Effect of pH and Salt on pDADMAC Capture of Prion Proteins

Background

The effects of pH and salt concentration on the effectiveness of captureof PrP^(Sc) to polycation-coated plates (formed by coating the wellswith poly(diallyldimethyl ammonium chloride) (pDADMAC), (AldrichChemical Company Inc., catalogue number 40,903-0) were investigated

Method

-   -   1. 80 μl of brain homogenate was made up to 100 μl by addition        of 20 μl of Capture buffer (250 mM Tris, see Table for pH, 5%        (v/v) Triton X-100, 5% (w/v) N-lauroyl sarcosine, 5% (w/v) BSA        and 6.25 mg/ml of Trypsin) containing various concentrations of        salt and adjusted to various pHs was investigated.    -   2. The homogenates were then added to polycationic-coated        microwells.    -   3. After incubation for 2 hours at room temperature, the wells        were washed six times with PBS.    -   4. 100 μl 4M Guanidine thiocyanate, 20% (w/v) PEG was added to        each well.    -   5. After incubation for 10 minutes at room temperature wells        were washed three times with PBS.    -   6. 100 μl of anti-prion protein horseradish peroxidase conjugate        (diluted 1:1500 in PBS 0.1% (v/v) Tween 20 and 5% (w/v) BSA) was        added.    -   7. After 1 hour at room temperature wells were washed five times        with PBS 0.1% (v/v) Tween20.    -   8. Immobilised conjugate was detected with TMB solution        following standard protocols and the OD450 of the reactions        measured.        Results

Effect of pH Capture Infected Negative bovine Buffer pH bovine brainbrain 5 0.177 0.119 6 0.082 0.1 7 0.093 0.045 8.4 0.226 0.039 9 0.240.038 10 0.25 0.037

Effect of Salt Infected Uninfected Capture bovine bovine Buffer brainbrain  20 mM 0.476 0.038 NaCl 100 mM 0.361 0.039 NaCl 250 mM 0.191 0.028NaCl 1M NaCl 0.06 0.024Discussion

As the pH of the Capture buffer is lowered the signal from theuninfected brain increases but the signal from the infected braindecreases. At pHs of greater than 8.0 the optimum positive to negativesignal ratio is achieved.

As the salt concentration in the Capture buffer is increased the signalfrom the infected brain progressively decreases. This indicates that alow salt concentration or no salt is the optimum condition for thePrP^(Sc) capture.

EXAMPLE 16 Effect of Varying Concentrations of N-lauroyl Sarcosine andProtease on pDADMAC Capture of PrPSc

Background

The effect of different N-lauroyl sarcosine concentrations in thepresence or absence of trypsin were investigated on the effectiveness ofcapture of PrPSc to polycation-coated plates (formed by coating thewells with poly(diallyldimethyl ammonium chloride) (pDADMAC), (AldrichChemical Company Inc., catalogue number 40,903-0) were investigated.

Method

-   -   1. 80 μl of infected brain homogenate was made up to 100 μl by        addition of 20 μl of Capture buffer (250 mM Tris pH 8.3, 5%        (v/v) Triton X-100, 5% (w/v) BSA) containing different        concentrations of protease and N-lauroyl sarcosine.    -   2. The homogenates were then added to polycationic-coated        microwells.    -   3. After incubation for 2 hours at room temperature, the wells        were washed six times with PBS.    -   4. 100 μl 4M Guanidine thiocyanate, 20% (w/v) PEG was added to        each well.    -   5. After incubation for 10 minutes at room temperature wells        were washed three times with PBS.    -   6. 100 μl of anti-prion protein horseradish peroxidase conjugate        (diluted 1:1500 in PBS 0.1% (v/v) Tween 20 and 5% (w/v) BSA) was        added.    -   7. After 1 hour at room temperature wells were washed five times        with PBS 0.1% (v/v) Tween20.    -   8. Immobilised conjugate was detected with TMB solution        following standard protocols.

Results Detergent and protease used in Concentrations of Capture Bufferagent used OD450 N-lauroyl sarcosine 0 0.08 Trypsin 1.25 mg/ml N-lauroylsarcosine  2.5% 1.181 Trypsin 1.25 mg/ml N-lauroyl sarcosine   5% 2.267Trypsin 1.25 mg/ml N-lauroyl sarcosine 10.0% 2.628 Trypsin 1.25 mg/mlN-lauroyl sarcosine 0 0.171 Trypsin 6.25 mg/ml N-lauroyl sarcosine  2.5%2.384 Trypsin 6.25 mg/ml N-lauroyl sarcosine   5% 2.725 Trypsin 6.25mg/ml N-lauroyl sarcosine 10.0% 2.883 Trypsin 6.25 mg/mlDiscussion

In the absence of N-lauroyl sarcosine there is no signal from theinfected brain with low concentrations of trypsin. At higherconcentrations of trypsin, however, some signal is restored in theabsence of N-lauroyl sarcosine.

Whilst the invention has been described with particular reference topreferred embodiments thereof it will be appreciated that manymodifications and variations thereof are possible within the generalscope of the invention. Any variation of the invention as explicitlyclaimed which would operate in the same way to produce the same resultis to be within the protection conferred by the application.

In this specification, unless expressly otherwise indicated, the word‘or’ is used in the sense of an operator that returns a true value wheneither or both of the stated conditions is met, as opposed to theoperator ‘exclusive or’ which requires that only one of the conditionsis met. The word ‘comprising’ is used in the sense of ‘including’ ratherthan in to mean ‘consisting of’.

1. A process for the selective binding of an aggregating abnormal formof a protein in the presence of the non-aggregating normal form of theprotein, comprising contacting under selective binding conditions amaterial containing both said abnormal and normal forms with a bindingagent which is a polyionic material having a binding avidity for saidaggregating form of said protein as present in the sample.
 2. A processas claimed in claim 1, wherein said selective binding conditions includethe presence of a competition agent in solution, which competition agenthas ionic groups having a lesser binding avidity for the abnormal formof the protein than does the polyionic material.
 3. A process as claimedin claim 1, wherein the binding agent is protease resistant.
 4. Aprocess as claimed in claim 1, wherein a protease is present during saidbinding or wherein said protein is exposed to the action of a proteaseafter being bound.
 5. A process as claimed in claim 1, wherein thebinding agent is a polyanionic material having a multiplicity of anionicgroups or a polycationic material having a multiplicity of cationicgroups.
 6. A process as claimed in claim 5, wherein said polyionicmaterial has a multiplicity of anionic groups which are sulphate,carboxyl or phosphate groups or a multiplicity of cationic groups whichare amino groups, imine groups or quaternary ammonium groups.
 7. Aprocess as claimed in claim 6, wherein the said polyionic material is apolyanionic polyglycoside.
 8. A process as claimed in claim 7, whereinthe polyanionic polyglycoside is a polysulphonated polyglycoside.
 9. Aprocess as claimed in claim 8, wherein the polyanionic polyglycoside isa polyanionic pentosan derivative or dextran derivative.
 10. A processas claimed in claim 9, wherein the poly-sulphonated polyglycoside ispentosan polysulphate (PPS) or dextran sulphate.
 11. A process asclaimed in claim 5, wherein said polyionic material is hexadimethrinebromide, PAMAM dendrimer, poly L-lysine, pDADMAC or polyethyleneimine.12. A process as claimed in claim 1, wherein the competition agent has alesser density of ionic groups than the polyionic material.
 13. Aprocess as claimed in claim 12, wherein the competition agent isanionic.
 14. A process as claimed in claim 13, wherein the competitionagent is an anionic detergent.
 15. A process as claimed in claim 13,wherein the competition agent is an amino acid amide of a fatty acid.16. A process as claimed in claim 15, wherein the competition agent isn-lauroylsarcosine.
 17. A process as claimed in claim 1, wherein the pHis such as to promote said binding of the binding agent to the abnormalform of the protein.
 18. A process as claimed in claim 17, wherein thepH is from 8 to
 9. 19. A process as claimed in claim 18, wherein the pHis from 8.2 to 8.6.
 20. A process as claimed in claim 1, wherein adetergent is present which promotes binding of the binding agent to theabnormal form of the protein.
 21. A process as claimed in claim 1,wherein said binding agent after binding to said aggregated abnormalform of the protein is captured with an immobilised capture agent.
 22. Aprocess as claimed in claim 21, wherein said capture agent is a lectinor an antibody reactive with said binding agent.
 23. A process asclaimed in claim 21, wherein the said binding agent is provided with aselectively bindable tag moiety and said capture agent binds to said tagmoiety.
 24. A process as claimed in claim 1, wherein the binding agentis immobilised to a solid medium prior to exposure to said abnormal formof the protein.
 25. A process as claimed in claim 24, wherein the mediumis a substrate having said binding agent coated thereon.
 26. A processas claimed in claim 24, wherein the binding agent is provided with aselectively bindable tag moiety and is immobilised to said solid mediumvia said tag moiety.
 27. A process as claimed in claim 23, wherein saidbindable tag moiety is biotin, fluorescein, dinitrophenol, digoxyrenin,a nucleic acid or nucleic acid analogue sequence or (His)₆.
 28. Aprocess as claimed in claim 1, wherein said binding agent in a solidwhich provides a surface having said binding avidity.
 29. A process asclaimed in claim 28, wherein the surface is that of a polymer havingionic groups covalently bonded within the structure of the polymer orproduced by modification of surface groups of the polymer.
 30. A processof assay for the presence of an abnormal aggregating form of a proteinin a sample, said process comprising the binding of said aggregatingform protein by contacting under selective binding conditions a samplecontaining said aggregating form protein with a binding agent which is apolyionic material having a selective binding avidity for saidaggregating form of said protein as present in the sample, followed bydetermining the existence or amount of binding of the protein to thebinding agent.
 31. A process as claimed in claim 30, wherein saidbinding is qualitatively or quantitatively determined by conducting animmunoassay for the aggregating form of the protein.
 32. A process asclaimed in claim 30, wherein the binding of the abnormal aggregatingform of the protein is conducted selectively in the presence of thenormal non-aggregating form of the protein and bound aggregated formprotein is then separated from non-bound normal form protein andthereafter said determination of the existence or amount of said bindingis determined.
 33. A process as claimed in claim 30, wherein saidabnormal form of a protein is PrP^(Sc) and said normal form is PrP^(C).34. A process for separating PrP^(Sc) from PrP^(C) comprisingselectively binding PrP^(Sc) to a binding agent in the presence of anamino acid amide of a fatty acid.
 35. A process as claimed in claim 34,wherein the binding agent is a polyionic material.
 36. A process asclaimed in claim 35, wherein the amino acid amide of a fatty acid isN-lauroylsacrosine.
 37. A process as claimed in claim 34, conducted at apH of from 8 to
 9. 38. A binding agent which is a polyanionicpolyglycoside, hexadimethrine, or a polycationic binding agentconjugated to a bindable tag moiety, whereby the binding agent can beimmobilised to a solid medium by the binding of said bindable tag moietyto a capture agent on said solid medium.
 39. A selective binding agentas claimed in claim 38, wherein the binding agent is a polysulphonatedpolyglycoside.
 40. A selective binding agent as claimed in claim 38,wherein the binding agent is a polyanionic pentosan derivative ordextran derivative.
 41. A selective binding agent as claimed in claim38, wherein the binding agent is a polycationic material selected fromhexadimethrine bromide, PAMAM dendrimer, poly L-lysine, pDADMAC orpolyethyleneimine.